The neurotrophic and growth-stimulating actions of vasoactive intestinal peptide (VIP) are mediated indirectly through secreted, glia-derived substances. Our focus is to characterize these VIP-mediators and study their mechanism of action. A principal glial mediator of VIPs neuroprotective actions is activity dependent neurotrophic factor (ADNF). Previous studies indicated that ADNF exhibited neuroprotection at femtomolar concentrations and had a structural similarity to heat shock protein 60 (hsp60). However, our recent studies indicate that ADNF preparations tested over a very broad range of concentrations exhibited two peaks of survival-promoting activity when tested in cerebral cortical cultures co-treated with tetrodotoxin. The EC50s of these activities differed by 100,000-fold. Although no ADNF heterogeneity was evident with five chromatographic techniques, analysis of purified ADNF by capillary electrophoresis (CE) demonstrated two peaks, each exhibiting survival-promoting activity, only one of which was blocked by antiserum to hsp60. Amino acid sequence data was obtained after trypsin digestion of ADNF. A total of 15.8 kDa was sequenced, with none of the peptides matching any known sequence. Antisera generated against one of the digest peptides produced neuronal cell death when incubated with cerebral cortical cultures and blocked the survival-promoting activity of ADNF and the non-hsp60 peak isolated by CE. These data suggest that ADNF is more complex than previously recognized and may consist of two active components, one sharing an epitope with hsp60, the other being a unique protein/subunit. To further explore the protective actions of VIP-related proteins, a peptide associated with ADNF and another peptide obtained from a related protein (activity dependent neuroprotective protein) were used to prevent embryonic death and growth restriction in a model of fetal alcohol syndrome. Furthermore, the protective actions of the peptides were effective even one hour after the administration of alcohol. VIP has a dramatic effect on mouse embryonic growth at the time of neural tube closure. In further investigations of the mediators of VIP-stimulated growth, isolated neural tube from embryonic day 9.5 mice were incubated with VIP and the conditioned medium (CM) studied. As analyzed by CE, 35 peaks were detected in CM. In this complex mixture, two cytokines were identified: interleukin-1 and interleukin-6. These cytokines have been shown to have proliferative and neurotrophic roles in the developing CNS. - VIP, PACAP, astrocytes, cytokines, stress proteins, neuroprotection, neural tube, fetal alcohol syndrome, trophic factors